CN112219757A

CN112219757A - Method for salmonization

Info

Publication number: CN112219757A
Application number: CN202011099200.9A
Authority: CN
Inventors: 吉姆·罗杰·努尔利; 阿瑟·里恩古伊; 克劳迪奥·雷塔马尔·曼西利亚
Original assignee: Stim Co Ltd
Current assignee: Stim Co Ltd
Priority date: 2014-09-23
Filing date: 2015-09-22
Publication date: 2021-01-15
Anticipated expiration: 2035-09-22
Also published as: DK3808188T1; EP3197290B1; WO2016046182A1; CN106998745B; CN106998745A; PL3197290T3; ES2836054T3; ES2728552T3; CN112219757B; SI3197290T1; PT3197290T; EP3398449A1; CA3148820A1; RS58906B1; RU2708168C2; PL3398449T3; EP3516965A1; CL2019000494A1; DK3398449T3; DK3398449T1

Abstract

The present application provides methods for salmoniding. A fish feed useful in a method for silvering and preventing desilvering in salmonidae and for preventing and treating Haemorrhagic Smolt Syndrome (HSS) in salmonidae. The feed contains protein, fat, carbohydrate, vitamins, minerals and water, and further comprises 10-100g/kg by weight of sodium salt (Na)⁺) 1-10g/kg by weight of a polyvalent cation receptor modulator (PVCR), 0.1-100g/kg by weight of a magnesium salt (Mg)²⁺) And 0.1-100g/kg by weight of calcium salt (Ca)²⁺)。

Description

Method for salmonization

The present application is a divisional application of chinese patent application No.201580051584.8 entitled "fish feed and method for silvering and preventing desilvering of salmonidae and for preventing and treating Hemorrhagic Smolt Syndrome (HSS) of salmonidae" filed on day 2015, 9, month 22.

Technical Field

The present invention relates to fish farming, in particular to fish farming of Salmonidae (Salmonidae species), more in particular to a novel fish feed and method for smoltification (smoltification) of Salmonidae and for preventing desilverization (desmolification) of Salmonidae as well as for preventing and treating Hemorrhagic Smolt Syndrome (HSS) of Salmonidae.

Background

Salmonella (Salmo sp.), Gelsemii (Onchoronchus sp.) and Coleoptera (Salvelinus sp.) are species belonging to the family Salmonidae, which have a life cycle of laying eggs dating. The life cycle of the river-tracing spawning means that the fish stays in fresh water and seawater in the life cycle.

Salmon in fresh water, when they decide to migrate into the sea, undergo a physiological process called silvering.

In nature, the silvering process is governed by endogenous processes within the fish, and these are all synchronized with external signals from the fish environment (examples are darkness, light, water temperature, etc.). Smolt is the name of salmon in fresh water, which is ready to migrate into the sea. The process of silvering involves several endocrine signaling substances, such as melatonin released by the pituitary gland, Thyroid Stimulating Hormone (TSH), Prolactin (PRL), Growth Hormone (GH), and adrenocorticotropic hormone releasing hormone (ACTH). These substances have an effect on several target organs in the fish body (examples are thyroid and adrenal glands), which secrete signaling substances that in turn alter the appearance, behavior, growth and metabolism, body composition and capacity to maintain osmotic balance in seawater.

In fresh water, salmon will release ions (Cl) from the environment^-、Na⁺、K⁺、Ca²⁺) Pumped into the body (e.g. by gills) to reabsorb ions from the urine (e.g. Ca)²⁺、Mg²⁺) And at the same time, strongly excretes dilute urine, thereby disposing of excess water in the body. When the fish acclimates to the sea, this physiological activity turns in the opposite direction. By the process of silvering, salmon becomes able to pump salt out of the body (e.g. by gill)Pump out Na⁺And Cl^-) Secretion of excess ions by the urine (example is Ca)²⁺、Mg²⁺) And reabsorbs water from the urine in the kidneys.

It is often observed that farmed salmon undergoes silvering in fresh water, with drowsy behavior, prominent skin scales, pale gills and bleeding from many internal organs (e.g. heart-and skeletal muscle, liver and visceral adipose tissue). Fish stocks may have a moderately increased mortality rate. This condition is known as hemorrhagic smolt condensation syndrome (HSS). The etiology of this disease is not fully understood. Possible explanations suggested in the scientific literature are malnutrition, genetic diseases and the presence of viral particles in tissues.

Smolt still remains in fresh water after the silvering process is complete and tends to develop a scaly skin (pine scales). The loose scales present a challenge in the handling and transport of fish, as they can easily cause skin lesions. In fresh and sea water, such lesions may become the entry point for infections (examples are saprolegnia, Moritella viscosa, Tenacibaculum maritimum) and cause ulceration and disturbed osmotic balance.

In the case where fish that reach the condition of smolt remain in fresh water, the silvering process will be reversed and the fish will attempt to reestablish a physiological balance suitable for living in fresh water. This process, known as desilverization, may be accompanied by decreased appetite, scaling and, oftentimes, a modest increase in mortality.

The use of traditional winter signals (12 hours light daily, 12 hours dark light manipulation) in salmon secondary-instar salmon production encounters a number of challenges. Winter signals reduced daily feeding and growth by 30%. A winter signal is provided for about 7 weeks, followed by a summer signal (24 hours light daily) until a smolt condition has been reached. In addition, pond sugar with high density of fish was observed in hatcheries with intensive production conditions (e.g. pond sugar)>70kg/m³) Causing the fish to receive different amounts of summer signals, which again causes the fish to silvery at different times. Fish living in the bottom of ponds with dark walls are more prone to receiving inadequate summer signals.

If not enough water treatment is achievedOr water exchange, too high biomass in the pond can adversely affect water quality (an example is increased levels of CO₂，>15 mg/l). Poor water quality can adversely affect the silvering process.

In addition, the big fish is silvered before the small fish, similar to desilverization. In hatcheries where conditions of production are intensified, maintaining fish of substantially similar size in each pond is a challenge due to the limited number of ponds available. Thus, fish populations often have fish of different sizes, whereby silvering and desilvering occur at different times in the fish population.

As the silvering process progresses, the fish are less and less able to stay in the fresh water, as their physiology adapts to the seawater. The occurrence of this phenomenon may lead to a modest increase in mortality among fish stocks. At this stage of production, fish with HSS are often found and reduced appetite and growth is observed.

In general, these conditions challenge fresh water and seawater production. In fresh water production, there is reduced growth and some mortality, while for seawater production, the transfer of smolt populations into the seawater has an uneven smolt status. This means that some fish in the sea die of osmoregulation problems, or that fish can survive but eat poorly and are more subject to long-term stress, followed by secondary disease. For a few years, the average mortality rate for norwegian salmon production has gone from the time of transfer to approximately 20% harvest. A survey conducted by the norwegian food safety agency (2013) showed that about 40% of the mortality rate was due to a reduction in the quality of smolt.

Organs involved in the process of silvering (e.g., pineal, hypothalamus, pituitary gland, kidney, intestine, gill, and skin) have a type of receptor known as calcium sensing receptors (CaSR) on the outside of the cell wall. The CaSR may be affected by different modulators, including ions (such as Ca)²⁺、Mg²⁺、Cl^-、Na⁺、H⁺) And free amino acids (such as tryptophan). Stimulation of CaSR provides up-regulation or down-regulation of the intracellular activity diversity of the cell. Controlled stimulation of the CaSR may provide a response corresponding to the silvering process.

An example of such controlled stimulation is

Method of reacting with a solution containing added Na⁺Ions, Cl^-Ion and Tryptophan Fish feed together with the ions added to the working Water (Ca)²⁺、Mg²⁺、Cl^-) In (1).

The process is described in international patent application WO 02/30182, the contents of which are "incorporated by reference" herein, as if written in their entirety in this application. And

the term "fish feed" as understood in connection with the method and further used in the present application is understood to mean a feed consisting of protein, fat, carbohydrates, vitamins and minerals, intended for smolt and smolt of salmon in freshwater. The composition of the growing feed may contain these or part of these materials. The skilled person is familiar with the type of feed to be used for this purpose, in contrast to feeds intended for other species or growth stages. In that

An example of fish feed for use in the method is shown in the product description shown in figure 28.

Under the condition that the winter signal and the summer signal (dark/light) are not used but continuous light (24 hours/day) is used until the time of transfer to the sea water,

the method makes it possible to silverise salmon, thereby avoiding the use of winter signals that reduce growth. In addition, using this method allows keeping the fish at the smolt window, preventing desilverization, which allows normal growth also during silvering in fresh water.

In the production of fresh water and in the production of seawater,

the method as a whole offers significant advantages in terms of production efficiency. The fish are kept in normal growth in fresh water and in seawater, and the smoothie population transferred into the sea has a uniform smoothie status, thus reducing production losses due to mortality, loss of appetite and increased risk of disease.

However, it is possible to use a single-layer,

the method has several disadvantages. This method requires the addition of large amounts of salt (ions) to the working water over a long period of time (3-6 weeks). This is a critical practical problem and adds significantly to the cost of production. Therefore, the method is rarely used to keep fish in fresh water for a long time<6 weeks) because of the cost and practical conditions that make it unsuitable.

Disclosure of Invention

Thus, according to one aspect, the object of the present invention is to provide a fish feed and elimination or reduction thereof

The disadvantages of the process. This is done by formulating a fish feed that is capable of silvering the fish alone without the use of salt in the process water, while allowing the fish to remain in the smolt window for an extended period of time. And

in contrast to the method, the present invention provides a simplified silvering process, since it does not require the addition of salts in the process water, and during silvering it can be easily implemented as an additional stimulus using winter and summer signals or insufficient winter and summer signals. In addition, it would be possible to keep salmon in fresh water without fish desilverizing, extincting or experiencing low growth rates until harvest size: (b)>200 grams, most often 4-6kg) are not experienced as hemorrhagic smolt syndrome or loose scales in the skin.

According to one aspect, the invention provides a method wherein the fish can be kept in fresh water until harvest size.

According to one aspect, the invention provides a fish feed comprising protein, fat, carbohydrate, vitamins, minerals and water, 10-100g/kg NaCl, with 1-10g/kg added multivalent cation receptor modulator (PVCR) (e.g. tryptophan or phenylalanine), further with 0.1-100g/kg added magnesium salt (Mg)²⁺) Such as MgCl₂And/or 0.1-100g/kg calcium salt (Ca)²⁺) For example CaCl₂。

Drawings

Figure 1 shows the development of the average Na + -K + -ATPase enzyme activity in the gill tissue of atlantic salmon fed test diet 1 and control feed a after winter signalling (n-24/sample in each group);

figure 2 shows the development of index of smolt in atlantic salmon fed test diet 1 and control feed a after winter signalling (n-24/sample in each group);

figure 3 shows the development of mean plasma chlorides in fish fed test diet 1 and control diet a in seawater (34% o, 96 hours) after winter signal (n 15/sample in each group);

fig. 4 shows the plasma magnesium and plasma calcium at

point measurement

11, 4 months 2012, using test diet 1 in field test 1, and compared to control diet a, with the results being the average values of fish with hemorrhagic smolt salmon syndrome (HSS) in the test (n-6) and control ponds (n-6), which values are compared to the average value of normal fish from the control group (n-6), the average value of normal fish in freshwater from field test 3 (n-19) and the reference value from literature (Jakobsen, 2013);

figure 5 shows the plasma chlorides at

point measurement

11, 4 months 2012, using test diet 1 in field test 1, and compared to control diet a, with the results being the mean values of fish with hemorrhagic smolt salmon syndrome (HSS) in the test (n-6) and control ponds (n-6), which values are compared to the mean value of normal fish from the control group (n-6), the mean value of normal fish in freshwater from field test 3 (n-19) and the reference value from literature (jakob sen, 2013);

figure 6 shows the mortality in fresh water for the group receiving test diet 1, the increased mortality being due to hemorrhagic smolt syndrome compared to the group receiving control diet a (Halse, 2012);

fig. 7 shows the development of the average Na + -K + -ATPase enzyme activity in the gill tissue of rainbow trout (n 25/sample per group) after winter signal (12 hours light/12 hours dark) on receiving test diet 2 and control diet a from cages in chile's fresh water lake (4 test cages and 4 control cages) with graded removal of the smallest fish before delivery between 9 and 12 days 2012;

figure 8 shows the development of average Na + -K + -ATPase enzyme activity in gill tissues of atlantic salmon receiving test diet 2 and control diet a (n-30/sample in each group) when using continuous light, the test was from fresh water ponds, performed in triplicate experimental set-up;

figure 9 shows the development of mean α 1a mRNA expression in gill tissue of atlantic salmon using test diet 2 and control diet a (n-30/sample in each group), with the loss of the first sample and the second sample being 01.10.2012, when continuous light was used;

figure 10 shows the development of mean α 1a mRNA expression in the gill tissue of atlantic salmon fed test diet 2 and control diet a with continuous lighting (n ═ 30/sample in each group), both groups missing the first sample point, and the test group missing the second and third sample points;

figure 11 shows the proportion of samples with copy values below α 1a mRNA expression below 1186000 in gill tissue with continuous light, which correlates with water temperature, and which are atlantic salmon fed test diet 2 and control diet a (n-8-10/sample in each group, two test groups/control group);

figure 12 shows the development of the average smolt index in fish receiving test diet 2 and control diet a (n 30/sample per group/sample point) from ponds in fresh water in triplicate experimental set-up by continuous light;

fig. 13 shows a scatter plot of atlantic salmon showing plasma chloride (mmol/l) after exposure to seawater (34% o, 144 hours) of fish receiving test diet 2 and control diet a within 11 weeks, which correlates with weight (g), with sample material being n-30 for the test group and n-20 for the control group;

figure 14 shows plasma chlorides (mmol/l) in the fish after exposure to sea water (34% o, 144 hours) in atlantic salmon fed for 11 weeks on test diet 2 and control diet a in fresh water, with the sample material being n-30 for the test group and n-20 for the control group, showing reference values for fresh water plasma chlorides (n-17/test and n-19/control);

figure 15 shows the magnesium and calcium (mmol/l) in the blood plasma of the atlantic salmon fed test diet 2 and control diet a for 11 weeks in fresh water after exposure to seawater (34% o, 144 hours), with the sample material being n-30 for the test group and n-20 for the control group, and also the values of fresh water (n-17/test and n-19/control);

figure 16 shows the development of the average Na + -K + -ATPase enzyme activity in gill tissues of atlantic salmon fed on test diet 2 under natural light conditions relative to control diet b (n-20/sample point in each group);

figure 17 shows the development of mean α 1a mRNA expression in gill tissue of atlantic salmon fed on test diet 2 under natural light conditions relative to control diet b (n ═ 14/sample point in each group);

figure 18 shows the development of the mean smolt index for atlantic salmon fed on test diet 2 under natural light conditions relative to control diet b (n-20/sample point in each group);

FIG. 19 shows the average development of Na + -K + -ATPase enzyme in the gill tissue of Atlantic salmon fed test diet 2 and control diet b during winter signaling, with the number of samples per sampling point listed in Table 14;

FIG. 20 shows the development of average Na + -K + -ATPase enzyme activity in Atlantic salmon gill tissues fed test diet 2 and control diet b after winter signal;

figure 21 shows the development of the average Na + -K + -ATPase enzyme activity in the gill tissues of atlantic salmon fed test diet 2 and control diet b after winter signalling;

FIG. 22 shows the development of the average Na + -K + -ATPase enzyme activity in gill tissue in Atlantic salmon fed test diet 2 and control diet b after winter signal;

figure 23 shows the observed percentage of expression values below 1186000 copies of α 1a mRNA in gill tissue after winter signalling, which correlates with water temperature, for atlantic salmon fed test diet 2 and control diet b (n-6-10/sample in each of the three test groups);

figure 24 shows the development of the mean smolt index for atlantic salmon using test diet 2 and control diet b after winter signalling, see table 14, which gives the number of samples per sampling point;

figure 25 shows a comparison of the average Na + -K + -ATPase enzyme activity in the gill tissue of atlantic salmon, the test group receiving light and test diet 2 in conjunction, while the control group received classical light manipulation and control diet b (n-20/sample point in each group);

figure 26 shows the development of mean α 1a mRNA expression in the gill tissue of atlantic salmon, the test group received light and test diet 2 continuously, while the control group received classical light manipulation and control diet b, the control group received a summer signal from 12.09.13 (n-20/sample in each group);

figure 27 shows a comparison of the average smolt index in atlantic salmon, test group received continuous light and test diet 2, while control group received classical light manipulation and control diet b (n ═ 20/sample point in each group);

FIG. 28 is a drawing showing

Product sheet of prior art fish feed used in the process.

Detailed Description

According to one aspect, the present invention provides a fish feed supplemented with a salt (ions) according to table 1 below and a PVCR modulator (free amino acids). All numerical ranges specified should be considered to include the various intermediate ranges as if such intermediate ranges were explicitly mentioned, e.g., ranges 1-10 should be considered to also include 1-9, 1-8, 1-7 (etc.); 2-10, 3-10, 4-10 (etc.); 1-9, 2-8 (etc.).

TABLE 1

PVCR modulators include the free amino acids mentioned herein, either alone or in combination: tryptophan, tyrosine, phenylalanine, serine, alanine, arginine, histidine, leucine, isoleucine, aspartic acid, glutamic acid, glycine, lysine, methionine, proline, glutamine, asparagine, threonine, valine and cysteine at a concentration of 1-10g/kg fish feed.

According to another aspect, the fish feed may comprise various combinations of the above additional ingredients. Non-limiting examples of such combinations are:

na, Cl, Ca and Mg

2.Cl、Ca、Mg，

3.Ca、Mg，

4.Ca、Na、Cl

5.Ca、Na、Mg

6.Ca、Na

7.Ca、Cl

8.Ca

9.Mg、Na、Cl

10.Cl、Mg

11.Mg

12. One or more free amino acids, Na, Cl and Ca

13. One or more free amino acids, Na, Cl and Mg

14. One or more free amino acids, Na, Ca and Mg

15. One or more free amino acids, Na and Ca

16. One or more free amino acids, Na and Mg

17. One or more free amino acids, Cl, Ca, Mg

18. One or more free amino acids, Cl, Mg

19. One or more free amino acids, Cl, Ca

20. One or more free amino acids, Ca, Mg and Cl

21. One or more free amino acids, Ca and Mg

22. One or more free amino acids and Ca

By "fish feed" is herein understood a feed consisting of protein, fat, carbohydrates, vitamins, minerals, pigments and water, which is suitable for smolt and smolt of salmon in freshwater. The composition of the feed for growing may contain these or part of these materials:

protein source:

soy protein concentrate (e.g., SPC65), Soy protein (e.g., HiPro Soy), pea protein meal, sunflower meal, wheat gluten, corn gluten, horse/broad beans, turnip meal, lupin, poultry meal, meat and bone meal, blood meal, guar meal, microbial proteins (from fermentation of different substrates), algal proteins, shellfish meal in general, krill meal, krill hydrolysate, fish meal (from NVG herring, mackerel, bowled mullet, capelin, anchovy, menhaden, and the like).

Carbohydrate source:

wheat or other suitable carbohydrate sources known in the art.

Fat source:

fish oil (from NVG herring, mackerel, horse mackerel, antelope, capelin, anchovy, herring, etc.), rapeseed oil, and linseed oil.

Minerals and vitamins:

the current nutritional recommendations for smolt and smolt addition for salmon in fresh water were followed.

Pigment:

astaxanthin or other pigments known in the art.

The person skilled in the art is familiar with such feeds and the necessary compositions to calculate the growth of smolt and smolt giving salmon in fresh water.

The fish feed according to the invention surprisingly enables:

smolt (10-150g) producing dative oviposition salmon for transfer to seawater

Maintenance of normal ion balance and osmotic regulation of the river-tracing spawning salmon in fresh water, including but not limited to

Preventing desilverization of salmon in fresh water,

prevention and/or treatment of the Hemorrhagic Smoothie Syndrome (HSS) of dative oviposition salmon,

preventing and/or treating the squamous edema causing the loss of the scales.

Post-secondary-age salmon (>150g) produced in fresh water for transfer to river-dating oviposition salmon in seawater.

Production of river-tracing spawning salmon in fresh water to market size for consumption (>100 g, most often 5000g)

The parent for producing the river-tracing spawning salmon in fresh water, as opposed to the cultured eggs/roe.

According to one aspect, the invention comprises adding feed equivalent to the feed

The feed is combined with additional magnesium and/or calcium salts, without the use of any other additive

These salts are mixed as in the process while operating with water.

According to another aspect, the invention provides a method to:

smolt (10-150g) producing dative oviposition salmon for transfer to seawater

Maintenance of normal ion balance and osmotic regulation of the river-tracing spawning salmon in fresh water, thereby achieving effects including, but not limited to

Preventing desilverization of salmon in fresh water,

omicron prevents, cures or treats Hemorrhagic Smoothie Syndrome (HSS) of dative oviposition salmon,

preventing, curing or treating the edematous putridity that causes the loss of putridity.

The method comprises the following steps:

a. a fish feed for parr or smolt is provided comprising protein, fat, carbohydrate, vitamins, minerals and water, 10-100g/kg NaCl, with 1-10g/kg added multivalent cation receptor modulator (PVCR) (e.g. tryptophan or phenylalanine) and 0.1-100g/kg added calcium salt (Ca)²⁺) For example CaCl₂And/or 0.1-100g/kg magnesium salt (Mg)²⁺) Such as MgCl₂，

b. In fresh water or brackish water, the feed is administered to the fish according to appetite until silvering occurs,

c. the fish were transferred to seawater after silvering.

Alternatively, after silvering, the fish may be kept in fresh water, in which case the method comprises after steps a and b: :

d. after silvering has occurred, the fish are kept in fresh water,

e. the application of fish feed to the fish is continued until it reaches the required weight in fresh water and is suitable for human consumption, or until it has reached an age/weight suitable for the introduction of sexual maturity, which may provide fish eggs for the production of new fish or for consumption.

The present invention will be described in further detail with reference to the following examples.

Materials and methods

Biological material, environmental conditions and experimental apparatus

Six field trials were performed using species of Atlantic salmon (Salmo salar) and rainbow trout (Onchnhychus mykiss). These fish were initially vaccinated with an oil-based vaccine and appetite was restored after vaccination. The fish initially have an average weight of at least 40 grams and finally have a weight of at most 180 grams in fresh water. In 2012 and 2013 in norway and chile, the test diets were used under normal production conditions. The test diets were fed to the fish for at least 3 weeks and up to 11 weeks, when only in fresh water. Tables 2 and 3 show information on species, stage, lighting conditions, water temperature, fish number, fish size, test feed and experimental set-up.

Table 2: overview of field trial (field meal), lighting conditions, Water temperature and trial diet type

Table 3: overview of field test, Lighting conditions, Water temperature and test diet type

Composition of test diet

Fish feed is herein understood to be a feed consisting of protein, fat, carbohydrates, vitamins and minerals, which is suitable for smolt and smolt of salmon in fresh water.

Test diet 1 (vs.

Feed):

a. fish feed added with 7% NaCl

b. Fish feed containing 0, 4% L-tryptophan

Test diet 2 (corresponding to an embodiment of the fish feed according to the invention):

a. fish feed containing 6% NaCl

b. Is added with0,75％CaCl₂Feed for fish

c. With addition of 0, 25% MgCl₂Feed for fish

d. Fish feed containing 0, 4% L-tryptophan

Control diet:

a. growing feed for smolt and smolt AS produced by Skretting AS

b. Feed produced by Ewos AS for growth of smolt and smolt

Parameters for monitoring the efficacy of test and control diets

Samples were taken before the fish received the test/control diet and immediately before the fish were transferred to seawater. In addition, sampling is performed between the initial sampling and the end sampling.

Na in gill tissue⁺-K⁺-ATPase enzymatic Activity

Samples were taken before the fish were fed the test diet and before the fish were transferred to seawater. Sampling is also performed to varying degrees between the initial and end point samples.

During the silvering process, Na was observed in gill tissue⁺-K⁺An increase in the amount of ATPase enzyme is normal. The main function of this enzyme is to pump salt from the fish, which is necessary to maintain osmotic balance in seawater. Gill tissue from a second gill arch was transferred to tubes and immediately frozen in liquid nitrogen (-180 ℃) in order to subsequently analyze the amount of gill enzyme in fisherguard AS, Leknes (formerly MultiLab AS) according to the method described in McGormick (1993).

Copy number of α 1a mRNA (fresh water ATPase):

The primary function of the α 1a mRNA-encoded enzyme is to pump salt from fresh water into the fish. Adaptation to life in seawater means that the enzyme activity must be reduced and similar for gene expression. During the silvering process, the copy number of α 1a mRNA in fish gills decreases. Gill tissue from the third gill arch was transferred to tubes with mRNA for analysis of the copy number of α 1a mRNA according to the method developed by fisherguard AS (2013).

Index of second instar salmon

Through the silvering process, changes in the appearance of the fish (morphological changes) were observed. This change was measured with the help of a smolt index score and was based on a visual score of 1-4 for each parameter (silver in skin, smolt marker and black fin edge), see table 4. The smolt index score is the average of the scores of all three parameters. In the presence of Na for analysis⁺-K⁺-index of smolt was recorded simultaneously with gill tissue of ATPase enzyme.

TABLE 4 summary of index scores for smolt

Chloride in fish plasma in seawater test

When there is an opportunity to carry out the seawater test, the fish are sampled and transferred to 34% of seawater and kept therein for 96 hours during the silvering process. Blood samples were taken from the fish and analyzed for chloride ion (Cl-) content according to the method adopted in central laboratory (2012) of norwegian veterinary science institute. This is a method for determining whether salmon in fresh water is silvered. If the fish had normal osmotic pressure regulation in seawater (after 96 hours in 34% seawater) (chlorine level in plasma of 120-150mmol/l), this is an indication that the fish were in the window of smolt.

Ions in the plasma of fish in freshwater

Blood samples were collected from fish in fresh water for analysis of Ca, Mg and Cl in plasma by two field tests. The analysis was performed in a central laboratory (2012) of the norwegian institute of veterinary medicine.

Mortality in fresh and sea water

In fresh water, the percent mortality during the use of the test feed and the control feed was recorded until the fish were transferred to the sea water. The mortality rate in seawater was observed for the group given the control feed in fresh water versus the group given the test feed. Mortality after 30, 60 and 90 days was enrolled, as well as total mortality at harvest (one sea farm).

Statistics of

Portions of the material were statistically processed to determine whether the change between the two measurement points in the test group was statistically significant (p 0.05 and p 0.01). A similar check was performed on the control group.

Results

Field test 1

Na + -K + -ATPase enzymatic Activity in gill tissues

FIG. 1 shows Na in gill tissue in field test 1⁺-K⁺Development of ATPase enzyme, wherein test diet 1 is used in comparison with control diet a, a growing feed for juvenile fish produced by Skretting AS. The results are the average of the results from the samples from 4 test ponds (n-24/sample) and 4 control ponds (n-24/sample).

Between 19.03.12 and 12.04.12, ATPase (ATPase) in the control group had a significant change (p 0.05), while ATPase in the test group did not (p 0.05). Between 26.04.12 and 09.05.12, ATPase varied significantly in the control group (p 0.05 and p 0.01) at 99% confidence level, while ATPase varied significantly in the test group only at 95% confidence level (p 0.05). Table 5 provides an overview of the subject.

Table 5: ATPase changes were significant between the two sample points in one group (p 0.05 or p 0.01). Comparisons were then made between the control and test groups. Other comparisons between the sampling points in the control and test groups did not have such differences in the significance observed.

Copy number of alpha 1a mRNA (fresh water ATPase)

No samples were taken for analysis of the copy number of α 1a mRNA, fresh water ATPase.

Index of second instar salmon

Figure 2 shows the development of the index for smolt in field trial 1, where trial diet 1 was compared to a control diet a, a growing feed for juvenile fish produced by Skretting AS. The results are the average of samples taken from 4 test ponds (n-24/sample) and 4 control ponds (n-24/sample). Between 19.03.12 and 26.04.12, there was a significant change in the index of smolt in the control group (p 0.01), while there was no significant change in the index of smolt in the test group (p 0.01). Between 12.04.12 and 26.04.12, the index of smolt in the control group had significant changes (p 0.05 and 0.01), while the index of smolt in the test group did not change significantly (p 0.05 and 0.01). Table 6 provides an overview of the subject.

Table 6: the change in index of smolt between two sample points in one group was significant (p 0.05 or p 0.01). They were then compared between the control and test groups. Other comparisons between the sampling points in the control and test groups did not have this difference in the significance observed.

Chloride in blood plasma of fish in seawater challenge test

Figure 3 shows the development of plasma chloride in test 1 in real time with test diet 1 and compared to control diet a, which is a growth feed for young fish produced by Skretting AS. The results are the average of samples taken from 2 test ponds (n 15/sample) and 2 control ponds (n 15/sample).

Ions in fish plasma in fresh water

Figures 4 and 5 show a single point measurement 11. Month 4 2012, plasma magnesium, plasma calcium and plasma chloride in fresh water in field test 1, where test diet 1 was used and compared to control diet a. The results are the average of fish with Hemorrhagic Smolt Syndrome (HSS) in the test (n ═ 6) and control (n ═ 6) ponds. This was compared to the average of normal fish from the control group (n-6), the average of normal fish in fresh water from field experiment 3 (n-19) and the reference from the literature (jakob, 2013). No reference value for normal plasma calcium in atlantic salmon has been successfully found in the literature.

Mortality in fresh water

Mortality during the experiment in fresh water is shown in figure 6. The highest mortality rate of 4 months in 2012 was about 10. Fish have classical authposy found that it is compatible with hemorrhagic smolt salmon syndrome (HSS), including pale gill and pale gut, various ecchymoses bleeding in muscle, abdominal adipose tissue and gut (Halse, 2012).

Field test 2

Na + -K + -ATPase enzymatic Activity in gill tissues

FIG. 7 shows the development of Na + -K + -ATPase enzyme in gill tissue when using test diet 2 in field test 2, compared to control diet a of growing feed for young fish produced by Skretting as. The results are the average of samples from the test group (n 25/sample) in 4 cages of fresh water and the control group (n 25/sample) in 4 cages of fresh water.

Between sampling points 30.08.12 and 06.09.12, between 30.08.12 and 20.09.12, and between 30.08.12 and 27.09.12, ATPase in the test group had a significant increase (p 0.01), while the control group had no significant change in ATPase at 99% or 95% confidence intervals (p 0.01 and p 0.05). Between 06.09.12 and 12.09.12, a significant increase in ATPase was observed in the control over the 99% site interval one week later compared to the test group (p 0.01). Between 12.09.12 and 20.09.12, the ATPase in the control group had a significant decrease within the 99% confidence interval, while the ATPase in the test group did not change significantly. Table 7 provides an overview of the subject matter.

Table 7: ATPase changes were significant between the two sample points in one group (p 0.05 or p 0.01). They were then compared between the control and test groups. Other comparisons between the sampling points in the control and test groups did not have this difference in the significance observed.

Copy number of alpha 1a mRNA (fresh water ATPase)

Index of second instar salmon

There were no samples for index checking of smolt in this trial.

Chloride of fish plasma in seawater challenge test

The smolt hatchery had no opportunity to perform the seawater test and no samples were taken.

Ions in fish plasma in fresh water

Sample free of ions in fish plasma in fresh water

Mortality in fresh and sea water

Mortality in the dilute phase was normal. After 60 days in the ocean, the mortality rate was 0.62% for the fish given test diet 2 and 0.71% for the fish given control diet a in fresh water.

Field test 3

Na + -K + -ATPase enzymatic Activity in gill tissues

Figure 8 shows the average development of Na + -K + -ATPase enzyme in gill tissue compared to the control diet a using test diet 2 in field trial 3. The results are the average of the samples from 3 test groups of fresh water ponds (n 30/sample) and 3 control groups of fresh water ponds (n 30/sample).

Between sample points 11.09.12 and 12.10.12, 11.09.12 and 05.11.12, there was a significant increase in ATPase in the test group (p 0.05), while ATPase in the control group did not change significantly within the 95% confidence interval (p 0.05). Between 12.10.12 and 05.11.12, ATPase decreased significantly (p 0.05) within the 95% confidence interval in the control group, while the test group did not have a significant change. Table 8 provides an overview of the subject.

Table 8: ATPase changes between two sample points within a panel were significant (p 0.05 or p 0.01). These were compared between the control and test groups. Other comparisons between the sampling points in the control and test groups did not show any difference in the significance observed.

Copy number of alpha 1a mRNA (fresh Water ATPase)

In both assay 1 and assay 2, a sample was partially taken to analyze α 1a mRNA, the copy number of fresh water atpase. The results are shown in fig. 9 and 10. Parallel assay 1 showed that test diet 2 yielded 441000-501000 copies of α -1a mRNA (fresh water ATPase) after two weeks of use. This is significantly below the limit of 1186000 copies. For control diet a, we seen that fish expressed a large number of copies 12.10.12 of α 1a mRNA, after which the expression was down-regulated to the same level as test diet 2.

Between sample points 01.10.12 and 12.10.12, there was a significant increase in α 1a mRNA expressed in the control group (p 0.05), while the α 1a mRNA in the test group did not change significantly within the 95% confidence interval (p 0.05). Between 01.10.12 and 06.12.12, the α 1a mRNA copy number in the control group was significantly reduced (p ═ 0.01) within the 99% confidence interval, while the test group was not significantly changed. The same applies between 12.10.12 and 05.11.12 (within 95% confidence interval), between 12.10.12 and 06.12.12, and between 05.11.12 and 06.12.12. The test groups did not show any similar variation between sampling points. Table 9 provides an overview of the subject.

Table 9: the copy number of α 1a mRNA within a panel varied significantly between two sample points (p 0.05 or p 0.01). These were then compared between the control and test groups. Other comparisons between the sampling points in the control and test groups did not show any difference in the significance observed.

Parallel assay 2 showed that, starting from the second week of use (01.10.12), the control feed gave 1517000 to 786000 copies of α 1a mRNA (fresh water ATPase). For the first three samples, this was above the limit for seawater tolerance (set to 1186000 copies), while the last sample was below the limit. For trial diet 2, we seen that fish expressed low copy numbers of α 1a mRNA, 05.11.12 and 06.12.12, between 413000 and 396000 copies. Fig. 10 provides an overview of the results.

There was a significant decrease in α 1a mRNA copy number expression in the control group between sample points 12.10.12 and 06.12.12 (p ═ 0.01). Between 05.11.12 and 06.12.12, the α 1a mRNA copy number in the control group did not decrease significantly (p ═ 0.01) within the 95% confidence interval, nor in the test group. Table 10 provides an overview of the subject.

Table 10: the copy number of α 1a mRNA varied significantly (p 0.05 or p 0.01) for both sample spots in one group. These were then compared between the control and test groups.

Water temperature-dependent copy number of α 1a mRNA (fresh water ATPase)

The α 1a mRNA due to the test group and the control group correlated with the fresh water temperature. Figure 11 shows the percentage of these samples that are below the threshold for seawater tolerance (1186000 copies of α 1a mRNA). The figure also shows the water temperature at the same time period. At water temperatures between 8.1 and 8.9 ℃, 90 and 100% of the sample values in the test groups are below the limit for seawater tolerance. This ratio is stable throughout the silvering process, in the same manner as the water temperature is stable. The corresponding values in the control group were between 20% and 100% and the lowest proportion was observed at the beginning of the observation period.

Index of second instar salmon

Fig. 12 shows the development of index for smolt using test diet 2 compared to control diet a, a growing feed for juvenile fish produced by Skretting as, in field test 3. The results are the average of the samples from 3 ponds (n 30/sample) in the test group and 3 ponds (n 30/sample) in the control group.

Between 11.09.12 and 01.10.12, the index of smolt in the test group increased significantly (p 0.01), while there was no significant change in the index of smolt in the control group (p 0.05). Between 01.10.12 and 12.10.12, the index of smolt in the control group increased significantly (p 0.05), while the index of smolt in the test group had a significantly robust increase (p 0.01). Between 12.10.12 and 06.12.12, the index of smolt in the control group increased significantly (p 0.01), while smolt in the test group did not change significantly (p 0.05). Table 11 provides an overview of the subject.

Table 11: index changes were significant between two sample points in one group (p 0.05 or p 0.01). These were then compared between the control and test groups. Other comparisons between the sampling points in the control and test groups did not have this difference in the significance observed.

Plasma chlorides in fish in seawater challenge test

Figures 13 and 14 show the plasma chloride status after exposure of the fish to 34% seawater for 144 hours in field test 3 when test diet 2 was used 11 weeks before seawater exposure, compared to control diet a. The results are the average of the samples from 3 ponds (n-30/sample) in the test group and 3 ponds (n-20/sample) in the control group. The mean value of plasma chloride in the control group was 139.6mmol/l, while the mean value of plasma chloride in the test group was 139.0 mmol/l.

Other ions in the plasma of fish in fresh and sea water

No bleeding smolt was observed in field trial 3. Figure 15 shows the average levels of magnesium and calcium in salmon plasma in fresh water and sea water.

Mortality in fresh and sea water

No abnormal mortality was observed. After 144 hours in seawater, the fish were destroyed.

Field test 4

Na + -K + -ATPase enzymatic Activity in gill tissues

Fig. 16 shows the development of Na + -K + -ATPase enzyme in gill tissue in field trial 4, using trial diet 2 compared to control diet b produced by Ewos AS growth feed for young fish. The results are the average of the sampled material from the test group freshwater cage (n 20/sample) and the control group freshwater cage (n 20/sample). The experiment was performed under natural light conditions, mainly after the autumn equinox.

Between 10.09.12 and 01.10.12, a significant change in ATPase was noted in the test group (p 0.01), whereas the ATPase of the control group did not change significantly during the same period (p 0.05). Between 10.09.12 and 15.10.12, the atpases in the test group had significant changes at 99% confidence intervals (p 0.01), while the atpases in the control group had no significant changes at 95% confidence intervals (p 0.05). Table 12 provides an overview of the subject.

Table 12: ATPase changes were significant between the two sample points in one group (p 0.05 or p 0.01). They were then compared between the control and test groups. Other comparisons between the sampling points in the control and test groups did not have this difference in the significance observed.

Copy number of alpha 1a mRNA (fresh Water ATPase)

Samples were taken for analysis of α 1a mRNA, fresh water ATPase copy number. The results are shown in FIG. 17. It shows that test diet 2 gave reduced expression of fresh water ATPase compared to control diet b. For trial diet 2, we seen a reduction in the copy number of α 1a mRNA from 259 to 67 tens of thousands, well below the limit of 1186000 copies. The difference between sampling points was significant within 99% confidence intervals (p ═ 0.01). The use of control diet b slightly reduced copy number from 259 to 257 ten thousand at the last sampling point. The drop between sampling points was not significant. Table 13 provides an overview of the subject.

Table 13: the copy number of α 1a mRNA in one group varied significantly between the two sample points (p 0.05 or p 0.01). They were then compared between the control and test groups.

Index of second instar salmon

Figure 18 shows the development of smolt index in trial 4 using trial diet 2 in real time compared to control diet b. The results are the average of the sampled material from the cages in the test group (n-20/sample) and the cages in the control group (n-20/sample).

Between 10.09.12 and 15.10.12, there was a significant increase in the index of smolt in the test group (p 0.05), while there was no significant change in the index of smolt in the control group during the same period (p 0.05). Table 14 provides an overview of the subject.

Table 14: the index of smolt in one group varied significantly between the two spots (p 0.05 or p 0.01). They were then compared between the control and test groups. Other comparisons between the sampling points in the control and test groups did not present this difference in the significance observed.

Chloride in fish plasma in seawater challenge test

No seawater challenge test was performed in the field test.

Ions in fish plasma in fresh water

There was no sample brought for analysis of the ions in the plasma while the fish were left in fresh water.

Mortality in fresh and sea water

No abnormal mortality in freshwater was observed. Fish that had received test diet 2 were previously labeled by sheared fat fins. The fish were transferred to the same cage in the sea as a light-manipulated group (unlike the fresh water control group). A mortality rate of 0.08% was observed from slaughtering of the fish. Mortality occurs immediately after exposure to seawater.

Field test 5

Na + -K + -ATPase enzymatic Activity in gill tissues

FIG. 19 shows the development of Na + -K + -ATPase enzyme in gill tissue when using test diet 2 in field test 5, compared to a growing feed control diet b for juvenile fish produced by EWOS AS. The results are the average of the sampled material from 3 ponds in the test group and 2 ponds in the control group. The number of ponds and fish per sample can be seen in table 15.

Table 15: in field test 5, an overview of pond number and fish number per sampling point.

Between 09.10.12 and 31.10.12, the increase in ATPase was more significant in the test group (p 0.01), while the increase in ATPase was less significant in the control group (p 0.05). Similar is true between sample points 20.11.12 and 04.12.12. Between 20.11.12 and 04.12.12, there was a significant increase in ATPase in the control group (p 0.05) within the 95% confidence interval, while there was no significant increase in ATPase in the test group (p 0.05) within the 95% confidence interval. Between 04.12.12 and 18.12.12, both groups had very similar p values (p ═ 0.01), but only the control group had a significant increase in ATPase within the 99% confidence interval. There was a significant increase between the two sampling points of the experimental group within the 95% confidence interval (p 0.05). In addition, we see that the ATPase of the experimental group had a significant increase between 12.12.12 and 18.12.12 (p 0.01), while the control group had no significant increase within the 95 and 99% confidence intervals (p 0.05 and 0.01). Table 16 provides an overview of the subject.

Table 16: ATPase changes were significant between the two sample points in one group (p 0.05 or p 0.01). They were then compared between the control and test groups. Other comparisons between the sampling points in the control and test groups did not present such differences in the significance observed.

Na + -K + -ATPase enzyme Activity in gill tissues in each parallel assay

FIG. 20 shows the development of Na + -K + -ATPase enzyme activity in gill tissue with test diet 2 in parallel assay 1 compared to control diet b.

Between 09.10.12 and 31.10.12, there was a significant increase in ATPase in the test group (p 0.01), while the ATPase in the control group was only significantly increased within the 95% confidence interval (p 0.05). Between sampling points 31.10.12 and 27.11.12 and between 20.11.12 and 27.11.12, there was a significant increase in the test group within the 99% confidence interval, while there was no significant increase in the control group. Table 17 provides an overview of the subject.

Table 17: ATPase varies significantly between two sample points in a group (p 0.05 or p 0.01). They were then compared between the control and test groups. Other comparisons between sampling points in the control and test groups did not show such significance.

FIG. 21 shows the development of Na + -K + -ATPase enzyme activity in gill tissue at the time of use of test diet 2 in parallel assay 2a compared to control diet b.

Between 09.10.12 and 31.10.12, there was a significant increase in ATPase in the test group (p 0.05), while the ATPase in the control group did not (p 0.05). Similar is true between sample points 20.11.12 and 04.12.12. Table 18 provides an overview of the subject.

Table 18: ATPase varies significantly between two sample points in a group (p 0.05 or p 0.01). They were then compared between the control and test groups. Other comparisons between the sampling points in the control and test groups did not show such differences in the significance observed.

FIG. 22 shows the development of Na + -K + -ATPase enzyme activity in gill tissue in parallel with 2b in the test diet 2 compared to the control diet b.

Between 20.11.12 and 18.12.12, ATPase in the test group had a significant increase (p 0.01) within the 99% confidence interval, while ATPase in the control group had a significant increase (p 0.05) within the 95% confidence interval. For sample points 12.12.12 and 18.12.12, the test group had a significant increase (p 0.05) within the 95% confidence interval, while the control group did not (p 0.05). Table 19 provides an overview of the subject.

Table 19: ATPase varies significantly between two sample points in a group (p 0.05 or p 0.01). They were then compared between the control and test groups. Other comparisons between the sampling points in the control and test groups did not show such differences in the significance observed.

Water temperature-dependent copy number of α 1a mRNA (fresh water ATPase)

Samples were taken only in the test group that received test diet 2. The copy number of α 1a mRNA (fresh water ATPase) was analyzed. Figure 23 shows the sample point ratio below the limit for seawater tolerance (1186000 copies of α 1a mRNA). The figure also shows the water temperature over the same period. In the first two weeks, the water temperature was above 6 ℃. After 2 weeks of feeding, 83 and 100% of the samples were below the limit of seawater tolerance. This percentage is further reduced by the silvering process, consistent with the reduced water temperature.

Index of second instar salmon

Figure 24 shows the development of the index for smolt using test diet 2 in field test 5, compared to control diet b. The results are the average of the samples from 3 ponds in the test group and 2 ponds in the control group. Table 14 gives an overview of the number of ponds and fish per sample point.

Between 20.11.12 and 12.12.12, the smolt salmon index in the control group had a significant increase (p 0.01) within the 99% confidence interval, while the smolt salmon index in the test group had a significant increase (p 0.05) within the 95% confidence interval during the same period. Between 04.12.12 and 12.12.12, the smolt salmon index in the control group had a significant increase within the 99% confidence interval (p 0.01), while the smolt salmon index in the test group did not have a significant increase within the 95% confidence interval (p 0.05). Table 20 provides an overview of the subject.

Table 20: the index of smolt in one group varied significantly between the two sample points (p 0.05 or p 0.01). They were then compared between the control and test groups. Other comparisons between the sampling points in the control and test groups did not show such differences in the significance observed.

Chloride of fish plasma in seawater challenge test

In this test, no seawater challenge test was performed.

Ions in fish plasma in fresh water

In this test, there was no sample for analyzing ions in plasma.

Mortality in fresh and sea water

No abnormal mortality was observed in fresh water. Meanwhile, no fish with disease HSS were observed. An overview of the mortality rate of seawater production is given in table 21 for ponds traceable to the test and control diets in seawater.

Table 21: mortality overview in seawater after transfer for a portion of the test materials in large freshwater

Field test 6

Na + -K + -ATPase enzymatic Activity in gill tissues

Fig. 25 shows the development of Na + -K + -ATPase enzyme in gill tissues when using test diet 2 in field test 6 compared to control diet b, which is a growth feed for young gills produced by Ewos AS. The results are the average of the samples from one cage (n 20/sample) in the test fresh water and one cage (n 20/sample) in the control fresh water. The test is a comparison of the two production methods, the fish in the test group receiving light and the test diet 2, while the fish in the control group received a combination of classical light manipulation (winter signal first, followed by summer signal) and the control diet b (regular growth drink).

The highest measured average ATPase value in the test group came about 5 weeks earlier than in the control group. A significant increase in ATPase was obtained in the test group after 2 weeks on the test diet (p ═ 0.01). The control group responded with a significant increase in ATPase 6 (p 0.01) 6 weeks after receiving the summer signal. Table 22 provides an overview.

Table 22: the variation in ATPase between two sample points in one group was significant (p 0.05 or p 0.01).

Copy number of alpha 1a mRNA (fresh Water ATPase)

Samples were taken for analysis of the copy number of α 1a mRNA (fresh water ATPase). The results are shown in fig. 26. It was shown that the combination of test diet 2 with continuous light provides lower expression of fresh water ATPase compared to control diet b and classical light manipulation. For test diet 2, we see a decrease in the copy number of α 1a mRNA from 607 ten thousand from the first sample to 75 ten thousand from the last sample, significantly below the limit 1186 ten thousand copies of seawater tolerance. The differences between the sampling points in the experimental groups were significant within 99% confidence intervals (p ═ 0.01) and were consistent with an increase in ATPase enzyme activity.

The use of control diet b increased copy number from 249 ten thousand for the first sample to 298 ten thousand for the last sample. The lowest average is reported at 23.10.13, 176 ten thousand copies, consistent with a significant increase in ATPase enzyme activity. The drop between 26.08.13 and 23.10.13 was within the 99% confidence interval (p 0.01), while the drop from 12.09.13 (which is the beginning of the summer signal) at 23.10.13 was not significant (p 0.05). Table 23 provides an overview of the subject.

Table 23: the copy number of α 1a mRNA varied significantly between two spots in one group (p 0.05 or p 0.01).

Index of second instar salmon

Fig. 27 shows the development of the smolt index in field trial 6. The results are the average of the samples from one cage (n-20/sample) in the test group of fresh water and one cage (n-20/sample) in the control group of fresh water.

There was a significant increase between index sampling points 26.08.13 and 23.09.13 for smolt in the test and control groups within the 99% confidence interval (p 0.01). The test group was transferred to seawater after 23.09.13, while the control group was transferred to seawater five weeks after the test group. During this period, several significant increases in smolt index were observed between the various sampling points in the control group. Table 24 provides an overview of the subject.

Table 24: the index of smolt in one group varied significantly between the two sample points (p 0.05 or p 0.01).

Chloride in fish plasma in seawater challenge test

In this test, no seawater challenge test was performed.

Ions in fish plasma in fresh water

In this test, there was no sample for analysis of ions in plasma.

Mortality in fresh and sea water

No abnormal mortality was observed in fresh water. Nor were fish with the disease HSS observed. Table 25 gives an overview of the mortality rate of seawater production.

Table 25: an overview of mortality from fresh water transfer to seawater.

Discussion of the related Art

Method selection and evaluation of silvering processes

It has been demonstrated in methods that the presence of CaSR in various organs associated with osmoregulation and endocrine activity is involved in the process of silvering, and it is known how the activity of these cells is influenced by the use of ions and amino acids that stimulate CaSR.

The method also provides Na⁺-K⁺Increase in ATPase enzymatic Activity, increase in index of smoltBehavior of second-instar salmon in fresh water, normal osmotic regulation in seawater (34 ‰), and good survival rate (1% mortality)<After 30 days) and growth in seawater production. All of these are conventional parameters for assessing whether a fish is satisfactorily silvered or not. From 2002 to 2014, there are over 30000 thousand super-silvered salmon, supporting that this method of adding salt to the working water by sampling can be used for the silvering process.

In assessing the effectiveness of the fish feed and method of the invention, a method has been applied and used to evaluate

Method of effectiveness similar method, which utilizes knowledge related to CaSR in combination with traditional silvering parameters.

In three of the six field trials, the test feed was used in combination with conventional light manipulation. In these cases, we must assume that the fish have endocrine activity corresponding to the normal silvering process. For the rest of the experiments, continuous or natural lighting was used after the autumn day and night, both conditions representing the challenge of obtaining a satisfactory silvering process and the ability to normally osmotically regulate, survive and grow after transfer to seawater.

When the term "silvering" is used in relation to the use of test diet 2, this means that endocrine activity in the test material is not examined, but is prone to changes in the parameters of conventional smolt. The nature of this work is therefore a practical approach to silvering in the production of smolt, not just in a complete survey of physiological factors associated with the actual silvering process.

Effect of

test diets

1 and 2 on the silvering Process in comparison to classical light manipulation

In field test 1, a combination of test diet 1 and ordinary photo manipulation was used, while in

field tests

2 and 5, a combination of test diet 2 and ordinary light manipulation was used.

Field test 1:

test diet 1 did not receive Na⁺-K⁺A significant increase in ATPase enzymatic Activity and similarity in control group. Similar results were observed for the increase in index for smolt. After 96 hours of seawater challenge test in 35% o of seawater, no significant change in plasma chloride was observed. Both the control and test groups were within the normal range of 120-150mmol/l of plasma chloride. However, on average, a 20-fold higher mortality rate associated with the disease HSS during silvering was observed for the fish receiving test diet 1 compared to the control group. For this size of fish, the test performed at water temperature 3-5 ℃ should provide an average dietary intake of 0.2-0.4% per day (Skretting feed table, 2009).

However, the feed intake was large enough to observe an increase in mortality in the test group, and as the water temperature increased, an increase in feed intake should be unlikely to benefit the survival of the test group. Overall, these observations provide a basis for demonstrating that test feed 1 alone is not a suitable diet for stimulating the silvering process in salmon. Test diet 1 was used

A kind of feed used in the method, but with Ca²⁺And Mg²⁺The combination is added to the working water.

Field test 5:

test diet 2 was used in field test 5. In this experiment, the water temperature was 8-6 deg.C (after the summer signal was given) during the first two weeks of the silvering process, and then the water temperature was first reduced to 4 deg.C and then 3 deg.C. The temperature drop is considered an environmental signal, which hinders the silvering process. Lowering the water temperature resulted in a decrease in feed intake, but it appears that the first two weeks with the highest water temperature and relatively high test diet 2 intake were critical to how the silvering process ran. At the early stages of the silvering process, the increase in ATPase enzyme activity between sampling points was significantly stronger in fish receiving test diet 2 (significance within the 99% confidence interval) compared to the control group (significance within the 95% confidence interval). The smolt index showed no corresponding increase in favor of test diet 2. Smolt index scores are somewhat subjective and may include variations in score between different samplers in the main hatchery. The sample material late in the silvering period in each group is also limited (n-10). The smolt index is rarely used to determine the time of transfer into the seawater, but more is an additional parameter in the silvering process.

Since the fish receiving test diet 2 satisfied smolt status, the test groups in replicates 1 and 2a were transferred to seawater 3 weeks and 2 weeks earlier than the control group, respectively. The test group in 2b was assayed in parallel for simultaneous transfer with the control group. However, based on ATPase values, the test groups could transfer into seawater 4 weeks earlier than the control fish.

The percent of α 1a mRNA (fresh water ATPase) in the test group with values below 1186 million copies increased significantly (about 2 weeks) from the start to the second sample point (fig. 23). The water temperature during this time was 6 ℃. As the water temperature decreased, the percentage of fish with α 1a mRNA (fresh water ATPase) below 1186 million copies decreased. This observation can be directly related to the feed intake of the fish, as a reduced water temperature will reduce the feed intake. At the same time, we see that test diet 2 gives additional stimulation of fish in the test group compared to the control group, shown as an additional boost in ATPase enzyme production.

In actual farming, late autumn transfers can be problematic relative to achieving satisfactory fish sizes in the sea before the winter. The fish is more prone to winter wounds than fish that come to the sea early in autumn. Early autumn transfers allow for greater utilization of higher seawater temperatures early in autumn, as well as achieving higher growth rates and reducing production time from transfer to slaughter. Late autumn transfers associated with decreased freshwater temperature and difficulties in the silvering process are common in the production of smolt. Trial diet 2 was a tool to achieve an earlier transfer time at reduced water temperatures in the fall.

Mortality in seawater at 30, 60 and 90 days post-transfer, respectively, was satisfactory for both the test and control groups. However, fish that had been given test diet 2 in fresh water had a lower mortality rate than the control group. The longer the time in the sea, the greater the difference in the percentage of mortality that occurs. Smolt status has an impact on survival in seawater and there is an increased risk of secondary problems associated with poor osmoregulation. In addition, these results support that test diet 2 is safe to use and does not negatively interfere with production.

Field test 2:

field test 2 was performed on rainbow trout. This fish receives winter signals in the southern hemisphere (before spring) in winter using natural light. After which additional illumination serving as a summer signal is received. Test diet 2 was used as an additional stimulus during the summer signal. It is not common to talk about the silvering process in rainbow trout, but the fact is that rainbow trout must respond with the same physiological response as salmon when transferred into seawater. Pre-adaptation in fresh water prior to transfer of fish to seawater seems to be a sensible strategy to reduce osmotic pressure regulation of rainbow trout. The test group showed an earlier significant increase in ATPase enzyme activity compared to the control group.

The hatchery has a program that grades the smallest fish in a fish school before delivery to the sea. This practice emphasises fish and results in a decrease in ATPase enzyme activity, confirming prior experience. Both the test and control groups responded with a decrease in the amount of ATPase following stress. However, only the control group had a decrease in ATPase, which was significant within the 99% confidence interval. The decrease in ATPase enzyme activity was not significant in the test group. The increase in ATPase in the control group was not significant the last week before transfer to seawater, but could be considered as a possible recovery after stress.

Rainbow trout was thinned (needles) the first time after transfer to the sea. The fish can survive in seawater for a long period of time, but eat poorly and do not grow normally. They are not easily removed from the cage; typically they go all the way to harvest time as they are produced. All fish were then counted and the real number of needle problems was shown. The disease state is not fully understood. However, it is assumed that poor osmotic regulation in seawater is a major factor. In this field test, the mortality rate after 60 days of transfer into seawater was satisfactory in both the test group and the control group, but the lowest mortality rate was observed in the test group.

Compared to the traditional production method, experimental trial 2 shows that trial diet 2 is a tool for better facilitating the pre-adaptation of rainbow trout to the life in seawater.

Effect of test diet 2 on the silvering process and the Effect of test diet 2 on desilvering without light manipulation

Field tests

3, 4 and 6 are experiments performed without light manipulation during the silvering process.

Field test 3

In field trial 3, fish placed in outside the hatchery were continuously exposed to artificial light. The fish were then moved indoors and continued to receive artificial light for 24 hours/day. Experiments were performed while the fish were indoors. Continuous light is generally not sufficient to achieve a satisfactory silvering process, but this condition, combined with high water temperature (>8 ℃), is known to provide high ATPase enzyme activity to fish in gills. Such fish may be normally osmotically regulated in seawater. However, these populations often exhibit heterogeneous smolt status within the population and are not suitable for transfer into the sea.

One aspect that may be important is that the fish catch eye in the dark night sky in august and september. This may be considered a winter signal, although a relatively modest amount of light from artificially added light is supplied in the pond compared to darkness in the night sky. The stimulus that has been perceived is considered to be a summer signal of the fish when transferred to an indoor hatchery and continuously supplied with stable light conditions without changes. As such, it aids in the silvering process. Regardless of the lighting conditions, it is well established that such fish have received a suboptimal light management solution compared to the standard for light management of smolt.

Test diet 2 provided earlier and higher ATPase enzyme activity compared to the control group. This was effective over the entire 11 week period of the experiment. There was a significant increase in the test group early in the silvering process (between the first and third sampling points), while the only significant change in the control group was a decrease in ATPase between the third and fourth sampling points. For test diet 2, there was a corresponding ATPase response as observed in

field tests

2 and 5.

In addition, we see a significant increase in the index of smolt in the test group between the first and second sampling points, while the control group did not have any significant change. Between the second and third sampling points, there was a more intense significant increase (within 99% confidence interval) in the test group compared to the control group (within 95% confidence interval). In this case, the increase in index of smolt in the test group occurred earlier than in the control group and was consistent with an increase in ATPase enzyme activity. An assessment of smolt index was made by the same person at each sampling time (except the first sampling) and the data material was 3 times greater than in field test 5 (n-30 compared to n-10). This underscores that test diet 2 had a significant effect on the smolt index in field test 5 when compared to test diet 2 lacking efficacy.

In the material used to analyze the copy number of α 1a mRNA (fresh water ATPase), we see that the assay ratios for amounts below 1186 million copies were nearly stable over the entire 11 week period of the experiment compared to the control group (fig. 11). Unfortunately, no sampling was initiated in the experiment, but the second sampling of replicate 2 indicated that the number of copies before the start of the experiment was high. Here we held the water temperature steady at 8.3-8.9 ℃ compared to field test 5 where the water temperature was reduced to below 6 ℃. The water temperature had an effect on the feed intake and the feed intake at 8 ℃ was stable, which was not achieved in field test 5. It is reasonable to assume that the percentage of stable samples with values below 1186 ten thousand copies of α 1a mRNA (limit for seawater tolerance) is of great significance. This result seems to be directly related to the intake of the test diet 2 and cannot be achieved by using the usual growth feed. This indicates that it can keep the fish at the smolt window while keeping for a longer time in fresh water. This has particular application in the production of actual smolt, the fact that the fish do not de-silvering, followed by synchronized smolt status throughout the fish population in the pond. This aspect is of great significance for growth and survival in seawater, but also provides flexible transfer time of smolt to seawater. Given suitable water temperature, water environment and good fish health, it is possible to provide post-bier salmon (1-2kg) in fresh water for delivery into marine facilities or for production of salmon in fresh water until harvest size (>2 kg).

We see that the control group passed the experimental period had a significant increase and decrease in fresh water ATPase, while the experimental group did not have any significant change. This may be due to the fact that the first sampling point for this type of analysis was missing in

replicates

1 and 2, and test diet 2 maintained the fish at a lower level of fresh water ATPase (fig. 9 and 10). However, after 11 weeks of the experiment, the level of fresh water ATPase in the control group was reduced to the same level as the test group. This may be because the fish have already entered the silvering process, which is about 654 days (assuming a summer signal is started) from the time the fish are moved into the hatchery compartment. Normally, the fish reached the smolt window after 350 days of summer signals. Comparing ATPase enzyme activity with fresh water ATPase at the same time period, we see ATPase enzyme activity of 7.7 in the control group and 9.2 in the test group at the last identical sample. The level of the control group indicates that desilverization, or that the fish are exposed to negative environmental influences and in this case a significant decrease in ATPase enzyme activity is not uncommon. In contrast, fresh water ATPase levels do not support desilverization. The most likely cause of reduced ATPase enzymatic activity is the stressor. Such stressors may have high density in small test ponds. This is similar to that observed when grading rainbow trout in field test 2, wherein the fish were fed test diet 2, maintaining a higher ATPase compared to the control group despite the addition of stressors.

In field trial 3, the fish were not transferred to seawater for further production. However, seawater challenge tests were performed prior to fish shoal destruction. This experiment shows satisfactory plasma chloride levels (120-150mmol/l) in the control and test groups (FIG. 14). We see that the trend in the material from the control group indicates to a large extent that the size of the fish positively affects the level of chloride in the plasma, in contrast to that observed in the test group (fig. 13). This observation supports that test diet 2 enhances the osmoregulation of fish in seawater.

Field test 4

The field test 4 was performed at a decreasing water temperature and a decreasing day length after autumn. The fish received longer darkness than the light exposure during the day and it was noted that the proportion of darkness per day increased during the test period. Both the water temperature and the day length of the decrease are negative signals for the silvering process. ATPase enzyme activity in fish fed test diet 2 had a significant increase within the 99% confidence interval, while the ATPase enzyme activity of the control group did not have any significant change. For test diet 2, these findings were similar to those observed in field test 3.

The smolt index of the fish fed test diet 2 had a significant increase between the first and last sampling, while the smolt index of the control fish was unchanged during the same period. Similar responses were observed in field trial 3 for trial diet 2.

For test diet 2, sample analysis of α 1a mRNA (fresh water ATPase) showed a significant increase in 99% confidence interval between sampling points. This decrease was observed in the control group. The second sample (about 2 weeks after initiation) in the test group was below the limit for seawater water tolerance (1186 copies of ATPase in fresh water). This response was observed in field test 3 as well as field test 5.

When transferred to seawater, mortality from the entire production is very low until harvest.

Overall, this experiment shows that test diet 2 is safe for use in normal production and it shows an effect on silvering, although there is no common silvering signal. In a real farming, the fish will receive a silvering signal that is often incomplete (lack/incomplete winter and summer signals) and in this case the test diet 2 can be used to compensate for this.

Field test 6:

the field test 6 was conducted to compare two different production methods, the combination of continuous light with the test diet 2 compared to conventional photographic operations and ordinary growing feed. This production takes place in the open air, in cages in fresh water. The fish in the test group received light continuously. In addition, the silvering process is completed before the autumn equinox. Thus, the fish obtain a longer day than night. The fish in the control group received natural light conditions until 9, 12 days 2012, and the night darkness during this period served as a winter signal. This will be perceived as a summer signal in the fish when exposed to artificial light. The main purpose of this experiment was to assess whether it was possible to transfer the fish into the sea at an earlier stage than possible photo manipulations. In addition, the effect of test diet 2 on silvering and the survival of fish after transfer into seawater were examined.

Experiments have shown that trial diet 2 is able to perform the transfer of smolt to seawater in this case, providing 5 weeks over light-operated smolt production. The main reason for this is that the fish do not get a winter signal and thus can maintain a normal feed intake. Thus, the fish reach the vaccination size earlier than fish receiving the winter signal. This second instar salmon factory cannot give an artificial winter signal because it is in the cage outside the freshwater lake. The second instar salmon must wait enough days to give darkness at night (in august and september) and after the winter signal, the artificial summer signal during silvering will be adjusted. ATPase activity had a significant increase in the 99% confidence interval between the first and second samples in the test group, whereas the ATPase of the control group did not have any significant change during the same period (fig. 25). For tests involving 2, this is a similar response observed in

field tests

3 and 4.

The smolt index had the same development in the test and control groups of this experiment. This may be due to the test and control groups receiving more natural sunlight than in the

field tests

3, 4 and 5. Light intensity is known to affect the extent of the index of smolt. Bright light results in a higher index of smolt than dim light.

Analysis of samples containing 2, α 1a mRNA (fresh water ATPase) for the trial showed a significant increase between sampling points within 99% confidence intervals. This drop also occurred between the first and second sampling in the control group, but the drop in the test group was numerically greater than the control group. This drop between the first and second samples in the trial group ranged from 607 to 148 ten thousand copies. Comparable, the control group decreased from 250 to 223 ten thousand copies. There were no significant changes in the control group between the second and third sampling points, but the test group continued to drop to 75 million copies. This change in the test group corresponded to that observed in

field tests

3, 4 and 5, but was somewhat delayed compared to the others. This may be due to the high number of α 1a mRNA copies initially observed in the experimental group, which takes longer to down-regulate this expression. When transferred to seawater, the control group did not reach below the seawater tolerance limit (1186 million copies of α 1a mRNA). It is known that the correlation between ATPase enzyme activity and α 1a mRNA in light-manipulated fish develops a u-shaped curve. From ATPase 1-9, mRNA copy number decreases, while from ATPase 9-22, more and more mRNA copy number occurs. It is possible that fish operate in a dual strategy in the region between 9-20 of ATPase and manage desilverization in fresh water and life in sea water. These lead to difficulties in finding the bottom level of fresh water ATPase at the time of sampling and this may also be the case in this experiment. At the same time, we see that the ATPase enzyme levels of the fish do not exceed 8,29 and this indicates that they are not ready to access seawater at the time of transfer. However, the control group showed good survival and growth in seawater. It can then be believed that ATPase enzyme activity is inhibited by the stressors and "released" when transferred into seawater.

The mortality rate of the fish fed test diet 2 in seawater for the first 8 months was very low, with a mortality rate of 0.36%.

Overall, this experiment shows that the test diet 2 is safe to use in normal production and that fish can be silvered without using photographic operations but with the aid of the test diet 2 only. However, combined with the use of continuous illumination

Experience with this type of production is limited compared to the use of the method. It is therefore natural to use caution, select favourable production conditions (high water temperature, healthy fish and good water quality) and use the method to gain a gradual experience before large scale use.

Physiological assessment related to Hemorrhagic Smolt Syndrome (HSS) in Atlantic salmon, and use of test diet 2 against HSS

HSS is a relatively common disorder during the silvering phase in atlantic salmon. Nylon et al (2003) correlated this disease with viral infection, but did not demonstrate a pathogen. It has also been suggested that malnutrition or genetic disease may be the likely cause (Rodgers and Richards, 1998). Typically, it is the largest fish in the fish population with HSS, and it is the farthest fish that is performed during the silvering process.

Physiologically, the smolt will actively pump Na in fresh water through the gills⁺And Cl^-And excretion of Mg by the kidneys²⁺And Ca²⁺. If salmon is in the smolt stage, it will absorb Na from the environment⁺、Cl^-、Ca²⁺And Mg²⁺. HSS problems arise when the fish are smolt acclimatised to seawater, but are still in fresh water and are further enhanced when the fish are fed an absorbent lining. Trial diet 1 resulted in an increased incidence of HSS by providing a 20-fold higher mortality rate than receiving normal growth feed. Test diet 1 included only Na⁺And Cl^-Without including free Mg²⁺And Ca²⁺. It is likely to be assumed that a NaCl content of 7% in the feed increases the drinking rate of fresh water for the fish and enhances the already established increased drinking rate in the fish population. For example, we see that HSS fish from field test 1 fed test diet 1 had a plasma chloride of 90,5mmol/l, whereas HSS fish fed the control diet had a plasma chloride of 102.8 mmol/l. The normal plasma chloride value of healthy fish in fresh water is 120-135 mmol/l. This indicates that the secretion of plasma chloride was amplified for fish with HSS feeding test diet 1 relative to fish with HSS feeding the growing feed. Plasma chlorides were significantly lower in HSS fish compared to normal fish, indicating an increase in Cl^-Is part of the pathological pattern. Probably for this reason, Na⁺-K⁺ATPase enzymatic activity stimulates fish by providing chloride ions, which fish obtain additional chloride ions added to the feed. HSS bearing fish with ATPase enzyme values are compatible with smolt status (ATPase of about 10 or more), and willThe good ability of the salt to be pumped out of the body is matched (MultiLab, 2012).

At the same time, as the fish actively excretes salt from the body on the kidneys and gills, the osmotic gradient between the fish and the water operates, causing water to flow into the fish and salt to flow out of the fish. Salt loss through osmotic gradients is advanced in the fish, which pumps out the salt even aggressively. In the kidney, fish are highly diuretic to separate out excess water, while reabsorbing ions such as Ca²⁺And Mg²⁺The ability of salmon is low, corresponding to the behavior of salmon in sea water. As a result, these ions are lost, which is a disadvantage when the fish is in fresh water. Thus, the fish enter states of hypocalcemia and hypomagnesemia. This is illustrated in fig. 4 and 15. The fish with HSS (test and control group) had 1.04 and 1.08mmol/l plasma magnesium, respectively. In the literature, the normal value for freshwater fish is 2mmol/l (Jakobsen,2013), while fish in field test 3 shows 1.44 mmol/l.

Similarly, for plasma calcium, the reference value for healthy fish from field trial 3 was 3.8mmol/l, while HSS fish (test and control) were 2.42 and 2.2mmol/l, respectively. Ca²⁺And Mg²⁺Is part of the fish's ability to perform normal muscle contractions. For other species such as cattle, it is known that hypocalcemia/hypomagnesemia can lead to muscle weakness, reduced heart rate and lethargy. The disease can be repaired by intravenous supply of calcium and magnesium. Looking at the clinical picture of fish in HSS you can observe the finding that sleepiness is typical. Fish swim slowly, which can be explained by muscle weakness (in the heart and skeletal muscles) due to hypocalcemia/hypomagnesemia. Empirical experience has shown that adding seawater to fresh water reduces or eliminates this type of death. Seawater is very rich in magnesium.

Another important autopsy finding in HSS is ascites (fluid in the abdominal cavity). If the heart muscle is unable to perform normal contractions and the heart rate drops, blood fluid is blocked in the vascular system. This may lead to exudates in the abdominal cavity of the fish, which we consider as ascites. In the case of fish lacking salt, the physiology is adapted to living in seawater, but in fresh water it will begin to drink to compensate for the loss of salt. This applies to Ca²⁺、Mg²⁺And Na⁺Or Cl^-. What is considered seawater (since it is smolt) is fresh water without salt. This makes drinking water non-obstructive and it gradually develops hypervolemia. Typically, autopsy, skeletal muscle edema, of fish with HSS was observed, indicating an excess of fluid in the peripheral circulation. Clinically, you can see prominent scales of fish, possibly due to skin/scale cavity edema caused by high blood volume.

Another important finding in autopsy of fish suffering from this condition is multiple blood stasis in the viscera and muscles. The blood vessels have smooth muscles, which are Ca-dependent²⁺And Mg²⁺To allow normal contraction. The lack of these ions results in a reduced contractile capacity and, in the case of hypervolemia, this may lead to vessel rupture and bleeding. In rainbow trout in seawater, impairment (blockage) of the pyloric sphincter function can lead to an imbalance in osmotic regulation, as the intestine cannot receive enough water from the stomach. This condition triggers the need for water and, to prevent drying, it starts drinking seawater. It will be consumed in an amount that causes a condition known as "ascites". This is an abnormally enlarged stomach filled with seawater. The condition may be in the range of abdominal muscle tears while the fish are still alive. This is similar to HSS, where salmon drinks fresh water to obtain salt to the extent that high blood volume is produced, which leads to vascular rupture and extensive bleeding in various organs. The exudate (ascites) that can be found by HSS, apparently due to a reduced pumping capacity of the heart to the incoming blood, can be enhanced by fish with symptoms of hypervolemia.

The fish feed according to the invention contains Na⁺、Cl^-、Ca²⁺And Mg²⁺And may be used in conjunction with silvering, the HSS occurring most commonly during silvering. The fish feed according to the invention may be used for the prevention and treatment of HSS conditions in salmon. By feeding the fish feed according to the invention, the fish does not need to drink fresh water to replace these ions mentioned herein, and therefore does not suffer from high blood volume, bleeding, ascites, muscle weakness or shell edema, and avoids HSS as a production problem.This would also apply to the production of large salmon in fresh water (until harvest size), where loss of appetite, HSS, shell edema and shell loss are common production hurdles.

Differences between the SuperSmolt method and the Fish feed according to the invention

According to

Method of adding a cation regulator Ca to process water²⁺、Mg²⁺And combined with Na in fish feed for salmon⁺、Cl^-And tryptophan.

The purpose of the method is to transfer salmon into sea water.

And

in contrast to the method, the fish feed according to the invention has all cationic conditioning agents in the feed itself and does not require separate addition of conditioning agents to the process water of the fish. The purpose of the fish feed and method of the invention is to transfer salmon into seawater, but also to keep the fish in a smolt window for long time production of large salmon in fresh water, and at the same time control disease HSS and desilverization. Thus, relative to

The fields of methods, fish feed, methods of silvering and applications are new.

In addition, we see that only the regulator Na is used in the feed⁺、Cl^-And tryptophan (e.g. in accordance with

The situation of the feed described in the method), the incidence of the disorder HSS in atlantic salmon is increased. This highlights the addition of the regulator Ca to fresh water without the use of this method²⁺、Mg²⁺In the case of (1), from

The diet provided by the feed is insufficient for a satisfactory silvering process.

The field of application of the fish feed according to the invention

The fish feed according to the invention can be used as:

1. silvered additional signal combined with traditional illumination manipulation in salmon.

2. Methods of silvering combined with continuous light, natural light, or incomplete light manipulation in silvering.

3. Groups of two-instar salmon were synchronized in fresh water.

4. Preventing the desilverization of the salmon in fresh water.

5. Preventing and treating HSS, edema and scale loss.

6. Feed composition for use in the production of post-mortem second-age salmon and harvest-sized fish in fresh water in various salmon species, in respect of preventing the occurrence of the diseases mentioned at

points

4 and 5 above, and maintaining normal growth similar to that seen in seawater.

7. Feed composition for producing parents in light of the various species of salmon until it is desired/possible to give a signal of sexual maturation and to harvest the size of fish eggs for consumption or for further production of fish, and for preventing the occurrence and maintenance of the diseases mentioned above at

points

4 and 5 similar to normal growth seen in sea water.

Claims

1. A method for salmonization, comprising the steps of:

a) a fish feed is provided comprising protein, fat, carbohydrate, vitamins, minerals and water, and 3.934-39.340 g/kg by weight of Na⁺0.026-25.530 g/kg Mg by weight²⁺And 0.036-36.110 g/kg Ca by weight²⁺And further comprising a multivalent cation receptor modulator (PVCR) in the form of a free amino acid at a concentration of 1-10g/kg by weight, and

b) in the ponds of the hatchery, the fish feed is administered to the smolt population for the purpose of inducing silvering until the fish reach a silvered state.

2. The method of claim 1, wherein the silvered fish of step b) is transferred to seawater.

3. The method of claim 1, wherein the feed is administered to the cohort of smolt while the cohort of smolt is in freshwater.

4. The method according to one of the preceding claims, characterized in that the free amino acids are selected from the group consisting of: tryptophan, tyrosine, phenylalanine, serine, alanine, arginine, histidine, leucine, isoleucine, aspartic acid, glutamic acid, glycine, lysine, methionine, proline, glutamine, asparagine, threonine, valine, cysteine, and any combination thereof.

5. Method according to one of the preceding claims, characterized in that Na⁺、Mg²⁺And Ca²⁺Are provided as salts in the ranges of 10-100g/kg, 0.1-100g/kg and 0.1-100g/kg, respectively.

6. The method of claim 5, wherein the sodium salt is NaCl.

7. The method of claim 5, wherein the magnesium salt is MgCl₂。

8. The method of claim 5, wherein the calcium salt is CaCl₂。

9. Method according to one of the preceding claims, characterized in that the fish feed comprises 6.202-199.020 g/kg Cl by weight^-。

10. Method according to one of the preceding claims, characterized in that the free amino acid is phenylalanine.

11. Method according to one of the preceding claims, characterized in that the free amino acid is tryptophan.

12. Method according to one of the preceding claims, characterized in that the fish feed comprises 6% by weight NaCl, 0.75% by weight CaCl₂0.25% by weight of MgCl₂And 0.4% by weight of L-tryptophan.

13. The method of claim 1, wherein the fish feed is administered to parr in conjunction with a schedule of continuous lighting conditions until silvering occurs.

14. The method according to claim 1, wherein the fish feed is administered according to the appetite of the fish and as a complete replacement for other fish feed.